Knockout of Pi21 by CRISPR/Cas9 and iTRAQ-Based Proteomic Analysis of Mutants Revealed New Insights into M. oryzae Resistance in Elite Rice Line

Abstract

<jats:p>Rice blast (Magnaporthe oryzae) is a devastating disease affecting rice production globally. The development of cultivars with host resistance has been proved to be the best strategy for disease management. Several rice-resistance genes (R) have been recognized which induce resistance to blast in rice but R gene-mediated mechanisms resulting in defense response still need to be elucidated. Here, mutant lines generated through CRISPR/Cas9 based targeted mutagenesis to investigate the role of Pi21 against blast resistance and 17 mutant plants were obtained in T0 generation with the mutation rate of 66% including 26% bi-allelic, 22% homozygous, 12% heterozygous, and 3% chimeric and 17 T-DNA-free lines in T1 generation. The homozygous mutant lines revealed enhanced resistance to blast without affecting the major agronomic traits. Furthermore, comparative proteome profiling was adopted to study the succeeding proteomic regulations, using iTRAQ-based proteomic analysis. We identified 372 DEPs, among them 149 up and 223 were down-regulated, respectively. GO analysis revealed that the proteins related to response to stimulus, photosynthesis, carbohydrate metabolic process, and small molecule metabolic process were up-regulated. The most of DEPs were involved in metabolic, ribosomal, secondary metabolites biosynthesis, and carbon metabolism pathways. 40S ribosomal protein S15 (P31674), 50S ribosomal protein L4, L5, L6 (Q10NM5, Q9ZST0, Q10L93), 30S ribosomal protein S5, S9 (Q6YU81, Q850W6, Q9XJ28), and succinate dehydrogenase (Q9S827) were hub-proteins. The expression level of genes related to defense mechanism, involved in signaling pathways of jasmonic acid (JA), salicylic acid (SA), and ethylene metabolisms were up-regulated in mutant line after the inoculation of the physiological races of M. oryzae as compared to WT. Our results revealed the fundamental value of genome editing and expand knowledge about fungal infection avoidance in rice.</jats:p>

Proteomics and Metabolomics Studies on the Biotic Stress Responses of Rice: an Update

“…Loss-of-function of Pi21 , a quantitative resistance gene encoding a proline-rich protein that includes a putative heavy metal-binding domain and putative protein-protein interaction motifs, results in non-race specific and durable blast resistance (Fukuoka et al 2009 ). Protein profiling of a Pi21 -knockout mutant in the absence of pathogen infection revealed the accumulation of photosynthates, carbohydrate metabolites, and small molecule metabolites, compared with the WT plants (Nawaz et al 2020 ). Additionally, metabolomics studies have shown that the enhanced basal resistance associated with loss-of-function of WRKY62 and WRKY76 (dsOW62/76) is associated with the accumulation of amino acids, constituents of TCA, phenolic acids derived from the phenylpropanoid pathway, upregulated SA and JA, and antimicrobial phytoalexins, such as sakuranetin and phenolamides (Liang et al 2017 ).…”

Precise Editing of the OsPYL9 Gene by RNA-Guided Cas9 Nuclease Confers Enhanced Drought Tolerance and Grain Yield in Rice (Oryza sativa L.) by Regulating Circadian Rhythm and Abiotic Stress Responsive Proteins

“…CRISPR/Cas9 can be used to precisely edit target genes to achieve the desired mutations and has higher mutagenesis efficiency and significance than other gene editing technologies [ 1 , 14 ]. CRISPR-Cas9 technology has been vastly used and made remarkable progress in the plant genome editing for improving agronomic traits, plant protection, grain yield, and abiotic stress tolerance [ 1 , 6 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ]. Recently, CRISPR-Cas9-guided SRL1 and SRL2 mutagenesis resulted in improved drought tolerance in rice at the seedling and mature stage [ 13 ].…”

CRISPR/Cas9 Directed Mutagenesis of OsGA20ox2 in High Yielding Basmati Rice (Oryza sativa L.) Line and Comparative Proteome Profiling of Unveiled Changes Triggered by Mutations

“…The targeted and effective gene mutations have been successfully achieved by the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system in rice for improving the existing varieties and to develop new mutant lines by targeting one or multiple genes [ 23 , 24 , 25 ]. CRISPR/Cas9 is a tremendous technique for precise and targeted editing of the genome of plants and animals.…”